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Le lymphome cutane primitif B diffus a grandes cellules type jambe (LBC-TJ), le plus agressif des lymphomes B cutanes, presente des caracteristiques proches des lymphomes B diffus a grandes cellules (LBDGC) de type « Activated B- Cell-like » (ABC) avec une activation constitutive de la voie NF-κB (nuclear factor-kappa B). Initialement, notre equipe a demontre la haute prevalence de la mutation MYD88L265P dans les LBC-TJ, puis associee a l’equipe du Pr Jardin (INSERM UMR1245, Rouen), nous avons confirme par exome sequencing la tres haute prevalence des mutations MYD88, PIM1 et CD79B [1] . La prise en charge de ces patients demeure un enjeu therapeutique malgre l’amelioration de la survie a 5 ans de 38 % a 74 % par l’association du Rituximab (anti-CD20) avec une polychimiotherapie adaptee a l’âge mais environ 50 % de non reponse sont observees sans facteur predictif connu. Nous souhaitons identifier un profil moleculaire predictif de reponse therapeutique permettant une meilleure prise en charge de ces patients. Nous avons analyse 14 patients repondeurs et 18 non repondeurs a la R-polychimiotherapie par NGS via un panel de 36 genes importants dans la lymphomagenese. Pour 14 patients, nous avons egalement analyse la biopsie a la progression/rechute afin d’identifier les alterations genetiques selectionnees lors de l’echappement therapeutique. Les patients presentant une mutation de la voie du BCR (CD79A/B ou CARD11) ont une survie sans progression et une survie specifique diminuee (p = 0,002 et p = 0,03 respectivement) [2] . L’analyse des echantillons au diagnostic et a la rechute montre que les mutations des genes MYD88 et CD79B sont les plus conservees et que la frequence allelique de ces mutations est superieure a la progression/rechute. La caracterisation d’une mutation dans la voie du BCR chez les patients atteints d’un LBTJ laisse presager une resistance a la polychimiotherapie conventionnelle. Ces patients pourraient beneficier d’un traitement adjuvant ou d’une seconde ligne d’emblee afin d’ameliorer leur prise en charge.

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF V600E and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAFV600E detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS). The presence of BRAFV600E mutation was assessed by at least two concordant techniques to further evaluate specificity and sensitivity of each method. The BRAF V600E mutation prevalence was detected in 18 out of 41 cases by ddPCR, 10 out of 36 cases by HRM PCR, and 16 out of 31 cases by NGS. BRAFV600E immunohistochemistry sensitivity was 94%, and specificity was 79%. HRM PCR sensitivity was only 59%, and specificity was 100%. NGS sensitivity and specificity were 100% for interpretable cases (n = 31), but in 11 cases, this technique was non-contributive. The analysis of BAL samples by ddPCR revealed a BRAFV600E mutation both in tissue and in BAL samples in one patient, a wild-type status both in tissue and in BAL samples in two patients, and a wild-type BRAF status in BAL and a BRAFV600E mutation in tissue samples in four patients. The study supports the usefulness of ddPCR for BRAF status assessment in either tissue or BAL samples to increase the accuracy of PLCH diagnosis.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

De recentes analyses de sequencage massif [1] , [2] ont permis de mettre en evidence la diversite et la complexite du profil mutationnel du syndrome de Sezary (SS). Ces differentes etudes ont cependant definit l’alteration de TP53 comme anomalie la plus frequente. Dans ce contexte, nous avons analyse le statut TP53 dans une cohorte de 35 patients SS ainsi qu’un groupe temoin compose de 8 patients presentant une dermatose inflammatoire chronique. Le statut TP53 a ete analyse a differents stades cliniques, notamment pour 9 patients appeles « SS secondaire » car ayant developpe un syndrome de Sezary apres un mycosis fongoides (MF). Une mutation du gene TP53 de type mutation ponctuelle ou petite insertion/deletion a ete caracterisee chez 10 (29 %) patients SS d’emblee ou secondaire. Aucune mutation n’est commune a plusieurs patients. A noter que ces mutations sont presentes a la fois dans les cellules mononuclees du sang et dans les echantillons de peau. Des analyses cytogenetiques realisees en parallele sur les cellules mononuclees du sang de 32 patients ont rapporte 27 (84 %) cas de deletion. Au total, une mutation et/ou une deletion de TP53 a ete caracterisee sur 29 (83 %) des 35 cas testes. Aucune difference de pronostique n’a ete observee selon le statut TP53 mais les patients presentant un SS secondaire ont un pronostic plus sombre que les patients presentant un SS d’emblee. Il est interessant de souligner que la presence de l’alteration de TP53 au diagnostic de SS (ou de MF s’il s’agit d’un SS secondaire) supporte l’hypothese que la mutation de TP53 jouerait un role pivot dans l’oncogenese du SS et du MF, et que l’etude du statut de TP53 et notamment l’absence d’alteration de TP53 pourrait etre utile au diagnostic differentiel des patients atteints de dermatoses inflammatoires.